Project 4: Genomics of TP53-mutated AML. The long-term goal of this project is to characterize mechanisms by which TP53 mutations contribute to AML progression and to exploit this knowledge to develop new therapies for this high-risk subset of AML. Mutations of TP53 are present in approximately 10% of de novo AML and 35% of therapy-related AML (tAML). Only ~25% of patients with TP53-mutated AML respond to standard induction therapy, and median survival is only approximately 4.2 months. Thus, there is a pressing unmet clinical need for new therapies for TP53-mutated AML. Due to the cytogenetic complexity and clonal heterogeneity of TP53-mutated AML, genetic and transcriptional alterations that contribute to its transformation and/or resistance to chemotherapy are largely unknown. In Aim 1, we will use single cell RNA sequencing along with phased whole genome sequencing to address this knowledge gap and to identify early transcriptional changes associated with the clonal expansion of TP53-mutated hematopoietic stem/progenitor cells (HSPCs). Our recent studies show that certain hematopoietic stressors, such as genotoxic stress or ribosome biogenesis stress, result in the selective expansion of hematopoietic stem progenitor cells (HSPCs) carrying heterozygous TP53 mutations. In Aim 2, we will test the following two hypotheses: 1) both cell-intrinsic factors and cell-extrinsic stressors contribute to the development of clonal hematopoiesis; and 2) repeated genotoxic or hematopoietic stress is important for the evolution from clonal hematopoiesis to AML. Specific Aim 1: We will identify genetic and transcriptional alterations associated with TP53-mutated AML. We will perform phased whole genome sequencing and comprehensive transcriptome sequencing, including single cell RNA sequencing, on a minimum of 50 fully annotated cases of aneuploid AML, 25 of which will carry TP53 mutations in the founding clone. These data will be analyzed to address the following questions: 1) is there a consistent pattern of gene dysregulation in TP53-mutated AML; 2) is there a consistent pattern of gene dysregulation in aneuploid AML compared to other AMLs; and 3) are there distinct structural alterations associated with TP53-mutated AML? We will perform single cell RNA sequencing on leukapheresis samples from patients with TP53-mutated clonal hematopoiesis to characterize gene expression in TP53- mutated HSPCs that do not carry other AML-relevant genetic alterations. Specific Aim 2: We will characterize the contribution of hematopoietic stressors to the development and progression of tAML/tMDS. We will use a mouse model of TP53-mutated clonal hematopoiesis to address three main questions: 1) what are the hematopoietic stressors that contribute to the expansion of TP53-mutated HSPCs or Dnmt3a-mutated HSPCs; and 2) what are the initial cooperating mutations that contribute to the clonal evolution of TP53-mutated HSPCs to tAML; and 3) what role does persistent or recurrent hematopoietic stress play in leukemic transformation?